1. Field of the Invention
This invention relates to a method for detecting and amplifying a target nucleic acid sequence in a sample. In particular, this invention relates to a polymerase chain reaction which is amenable to automation. With a polymerase chain reaction (PCR), one can "amplify" (i.e., increase the number of) target oligonucleotide molecules in a sample to be analyzed for the presence of that target sequence.
2. Description of Related Art
One type of polymerase chain reaction is disclosed in U.S. Pat. Nos. 4,683,202 and 4,683,195 which are incorporated herein by reference. In this type of polymerase chain reaction, two complementary polynucleotide strands are amplified by treating the strands with two oligonucleotide primers such that an extension product of each primer is synthesized which is complementary to each nucleic acid strand. The primers are selected such that the extension product of one primer forms a template for the synthesis of an extension product from the other primer once the extension product of the one primer is separated from the template. A chain reaction is maintained by a cycle of denaturing the primer extension products from their templates, treating the single-stranded molecule generated with the same primers to re-anneal, and allowing the primers to form further extension products. The cycle is repeated for as many times as it takes to increase the target nucleic acid segments to a concentration where they can be detected.
Typically, the amplified target sequence is detected by denaturing the double-stranded products formed by PCR, and treating those products with one or more reporter probes which hybridize with the extension products. The reporter probe has a detectable label, and is usually added in excess. The unhybridized reporter probe, therefore, must be separated from the hybridized reporter probe requiring a separation step. In another method of detecting the extension products without reporter probe and a separation step, the extension products are detected by gels stained with ethidium bromide.
Regardless whether reporter probes or gels are used, prior PCR methods are quite difficult to automate. Using reporter probes requires denaturing the extension products, annealing the reporter probe, and in some cases separating excess reporter probe from the reaction mixture. Using gels, of course, is time consuming, labor intensive, and thus impractical to automate if rapid results are desired.